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PAPRASTŲJŲ KARAGANŲ (Caragana Arborescens Lam.) LAPŲ FENOLINIŲ JUNGINIŲ KOKYBINĖS, KIEKYBINĖS SUDĖTIES IR ANTIOKSIDANTINIO AKTYVUMO TYRIMAS

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PAPRASTŲJŲ KARAGANŲ (Caragana Arborescens Lam.) LAPŲ FENOLINIŲ JUNGINIŲ KOKYBINĖS, KIEKYBINĖS SUDĖTIES IR ANTIOKSIDANTINIO AKTYVUMO TYRIMAS LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS FARMACIJOS FAKULTETAS NERINGA BIVEINYTĖ PAPRASTŲJŲ KARAGANŲ (Caragana arborescens Lam.) LAPŲ FENOLINIŲ JUNGINIŲ KOKYBINĖS, KIEKYBINĖS SUDĖTIES IR ANTIOKSIDANTINIO AKTYVUMO TYRIMAS Magistro baigiamasis darbas Vientisųjų studijų programa „Farmacija“, valstybinis kodas 6011GX003 Studijų kryptis „Farmacija“ Darbo vadovas Prof. habil. dr. Valdimaras Janulis KAUNAS, 2021 LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA FARMACIJOS FAKULTETAS FARMAKOGNOZIJOS KATEDRA TVIRTINU: Farmacijos fakultelo dekanė prof. dr. Ramunė Morkūnienė Data PAPRASTŲJŲ KARAGANŲ (Caragana arborescens Lam.) LAPŲ FENOLINIŲ JUNGINIŲ KOKYBINĖS, KIEKYBINĖS SUDĖTIES IR ANTIOKSIDANTINIO AKTYVUMO TYRIMAS Magistro baigiamasis darbas Darbo vadovas Prof. habil. dr. Valdimaras Janulis Data Recenzenzentas Dr. Vilma Gudienė Data Darbą atliko Magistrantė Neringa Biveinytė Data KAUNAS, 2021 3 TURINYS SANTRAUKA ..................................................................................................................................... 5 SUMMARY ......................................................................................................................................... 6 SANTRUPOS....................................................................................................................................... 7 ĮVADAS ............................................................................................................................................... 8 DARBO TIKSLAS IR UŽDAVINIAI ................................................................................................. 9 1. LITERATŪROS APŽVALGA ................................................................................................... 10 1.1. Karaganų (Caragana Lam.) genties augalų apibūdinimas, morfologiniai požymiai, paplitimas..................................................................................................................................... 10 1.2. Paprastųjų karaganų lapų, žiedų, šaknų ir sėklų cheminės sudėties tyrimai ............................... 12 1.3. Paprastųjų karaganų žaliavų panaudojimas medicinoje ir biologiškai aktyvių junginių poveikio tyrimas ................................................................................................................................................ 14 1.4. Fenolinių junginių ekstrakcijos iš augalinių žaliavų ėminių metodai ......................................... 16 1.5. ESC metodo taikymas fenolinių junginių analizei ...................................................................... 18 1.6. Spektrofotometrijos metodo taikymas fenolinių junginių analizei .............................................. 19 1.7. Antioksidantinis aktyvumas ir jo nustatymo metodai ................................................................. 20 1.8. Literatūros apžvalgos apibendrinimas ......................................................................................... 21 2. TYRIMO METODIKA .............................................................................................................. 22 2.1. Tyrimo objektas ........................................................................................................................... 22 2.2. Naudoti reagentai ......................................................................................................................... 22 2.3.Naudota aparatūra ........................................................................................................................ 23 2.4. Paprastųjų karaganų lapų ėminių ištraukų paruošimas................................................................ 23 2.5. Tyrimo metodai ........................................................................................................................... 24 2.5.1. Bendro fenolinių junginių kiekio nustatymas Folin – Ciocalteu metodika .......................... 24 2.5.2. Bendro hidroksicinamono rūgšties darinių kiekio nustatymas ............................................. 25 2.5.3. Bendro flavonoidų kiekio nustatymas .................................................................................. 26 2.5.4. Antioksidantinio aktyvumo įvertinimas ................................................................................ 26 4 2.5.5. Fenolinių junginių kiekybinis ir kokybinis nustatymas efektyviosios skysčių chromatografijos metodu ............................................................................................................................................. 28 2.5.6. Tyrimo metu gautų duomenų statistinis vertinimas .............................................................. 29 3. REZULTATAI IR JŲ APTARIMAS ......................................................................................... 30 3.1. Paprastųjų karaganų (Caragana arborescens Lam.) lapų ėminių fenolinių junginių ekstrakcijos sąlygų nustatymas ............................................................................................................................... 30 3.2. Paprastųjų karaganų lapų ėminių fenolinių junginių kiekio nustatymas ..................................... 32 3.3. Paprastųjų karaganų lapų ėminių hidroksicinamono rūgšties darinių kiekio nustatymas ........... 33 3.4. Paprastųjų karaganų lapų ėminių bendro flavonoidų kiekio nustatymas .................................... 35 3.5. Paprastųjų karaganų lapų ėminių ištraukų antioksidantinio aktyvumo in vitro nustatymas ....... 36 3.6. Fenolinių junginių kokybinės ir kiekybinės sudėties nustatymas ESC metodu paprastųjų karaganų lapų ėminiuose .................................................................................................................................... 38 3.7. Rezultatų apibendrinimas ............................................................................................................ 44 4. IŠVADOS .................................................................................................................................. ..45 5. PRAKTINĖS REKOMENDACIJOS ......................................................................................... 46 6. LITERATŪROS ŠALTINIAI .................................................................................................... 47 PRIEDAI ............................................................................................................................................ 52 5 SANTRAUKA N.Biveinytės magistro baigiamasis darbas/ mokslinis vadovas prof. habil. dr. Valdimaras Janulis; Lietuvos sveikatos mokslų universiteto, Medicinos akademijos, Farmacijos fakulteto, Farmakognozijos katedra, Kaunas. Pavadinimas: Paprastųjų karaganų (Caragana arborescens Lam.) lapų fenolinių junginių kokybinės, kiekybinės sudėties ir antioksidantinio aktyvumo tyrimas. Tyrimo tikslas: nustatyti paprastųjų karaganų (Caragana arborescens Lam.) lapų ėminių fenolinių junginių kokybinę, kiekybinę sudėtį ir lapų ištraukų antioksidantinį poveikį in vitro. Uždaviniai: Nustatyti tinkamas fenolinių junginių ekstrahavimo iš paprastųjų karaganų lapų ėminių sąlygas; įvertinti paprastųjų karaganų lapų ėminių fenolinių junginių įvairavimą vegetacijos periodu; nustatyti paprastųjų karaganų lapų ėminių ištraukų antioksidantinį aktyvumą in vitro, taikant UV regimosios šviesos spektrofotometrijos metodą; efektyviosios skysčių chromatografijos metodu nustatyti ir įvertinti fenolinių junginių kokybinę ir kiekybinę sudėtį karaganų lapų ėminių ištraukose. Tyrimo objektas ir metodai: paprastųjų karaganų lapų ėminiai, surinkti 2020 metais augalo vegetacijos periodu. Bendras fenolinių junginių kiekis ir antioksidantinis aktyvumas in vitro nustatytas UV regimosios šviesos spektrofotometriniu analizės metodu. Efektyviosios skysčių chromatografijos metodu nustatyta kiekybinė ir kokybinė fenolinių junginių sudėtis. Tyrimo rezultatai ir išvados: Nustatyta, kad didžiausias fenolinių junginių kiekis išgaunamas ekstrahuojant paprastųjų karaganų lapų ėminius su 40 proc. (v/v) etanoliu, taikant 20 min. trukmės ekstrahavimą ultragarso vonelėje, kai jos galia 1130 W, dažnis 80 kHz. Bendras fenolinių junginių kiekis lapų ištraukose įvairuoja nuo 15,65±0,67 mg GRE/g, p<0,05 iki 23,22±0,59 mg GRE/g, p<0,05. Didžiausias bendras fenolinių junginių kiekis lapuose nustatytas augalo vegetacijos periodo pradžioje ir iki augalo vegetacijos periodo pabaigos mažėja. Taikant ABTS metodiką stipriausias antiradikalinis aktyvumas – 66,78±28,35 µmol TE/g, <0,05; taikant FRAP metodiką stipriausias redukcinis aktyvumas – 95,65±7,86 µmol TE/g, p<0,05. Stipriausias antiradikalinis ir redukcinis aktyvumas paprastųjų karaganų lapų ėminiuose nustatytas liepos mėnesį, augalo žydėjimo laikotarpiu. Taikant ESC metodą, identifikuoti šie fenoliniai junginiai: kemferolis, izoramnetinas, kemferol-3-O-gliukozidas, hiperozidas, galanginas ir pinocembrinas. 6 SUMMARY Final Master‘s thesis by N.Biveinytė/ scientific supervisor prof. habil. dr. V.Janulis; Lithuanian University of Health Sciences, Academy of Medicine, Faculty of Pharmacy, Department of Pharmacog- nosy, Kaunas. The title of the master thesis: Qualitative and quantitative composition variability and antiox- idant activity of phenolic compounds in siberian peashrub (Caragana arborescens Lam.) leaves. The aim of the research: To determine the qualitative and quantitative composition of phe- nolic compounds and to evaluate the antioxidant activity in vitro of siberian peashrub (Caragana arbo- rescens Lam.) leaves. The objectives of the research: To determine the appropriate extraction conditions of phenolic compounds
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